Integrins are a family of cell adhesion receptors that mediate cell-extracellular matrix interaction and cell-cell interaction (Hynes, Cell 110(6): 673-87, 2002). It has been proposed that signaling from inside cells regulates the ligand-binding affinity of integrins (inside-out signaling) (Shattil, Trends Cell Biol 15(8): 399-403, 2005). Each integrin is a heterodimer containing α and β subunits. At present 18 α and 8 β subunits have been identified, which combine to form 24 integrins (Takada et al., Genome Biol 8(5): 215, 2007).
It has been reported that integrin αvβ3 plays a role in cancer proliferation and invasiveness. High levels of integrin αvβ3 correlate with growth and/or progression of melanoma (Albelda, EXS 61: 188-192, 1992; Hsu et al., Am J Pathol 153(5): 1435-42, 1998) neuroblastoma (Gladson et al., Am J Pathol 148(5): 1423-34, 1996), breast cancer (Pignatelli et al., Hum Pathol 23(10): 1159-66, 1992; Sengupta et al., J Exp Clin Cancer Res 20(4): 585-90, 2001), colon cancer (Vonlaufen et al., Mod Pathol 14(11): 1126-32, 2001), ovarian cancer (Liapis et al., Diagn Mol Pathol 5(2): 127-35, 1996), and cervical cancer (Chattopadhyay and Chatterjee, J Exp Clin Cancer Res 20(2): 269-75, 2001). Moreover, individuals homozygous for the β3L33P polymorphism that enhances the affinity of β3 integrins have an increased risk to develop breast cancer, ovarian cancer, and melanoma (Bojesen et al., Endocr Relat Cancer 12(4): 945-52, 2005). It remains unclear, however, if and how increased levels of αvβ3 on tumor cells contribute to cancer development.
Insulin-like growth factor-1 (IGF1) is a polypeptide hormone (75-kD) that has a high degree of structural similarity to human proinsulin. IGF1 acts through binding to the type I IGF receptor (IGF-IR), a receptor tyrosine kinase. The IGF-IR is a heterotetramer that consists of two α-subunits, which contain the ligand binding domains, and two β-subunits, which possess the tyrosine kinase activity. After ligand binding, the receptor undergoes a conformational change resulting in the activation of the tyrosine kinase, which leads to transphosphorylation of the opposite β-subunit on specific tyrosine residues. These phosphotyrosines then bind to adaptor molecules such as Shc and IRS-1. Phosphorylation of these proteins leads to activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (reviewed in Clemmons and Maile, Mol Endocrinol 19(1): 1-11, 2005).
IGF1 has been implicated in cancer progression (Clemmons et al., Growth Horm IGF Res 17(4): 265-70, 2007). One of the major actions of IGF1 is to inhibit apoptosis. IGF1 confers resistance to chemotherapy and radiation therapy. IGF1 expression levels are increased in breast, lung, prostate, and many other cancers. Several strategies to target IGF1 signaling have been extensively studied, including siRNA or monoclonal antibodies for IGF-IR and kinase inhibitors to inhibit the enzymatic activity of the receptor. The IGF1 system is a therapeutic target for cancer, and elucidation of the IGF1 signaling pathway should have a major impact in designing new therapeutic strategies.
It has been proposed that ligand occupancy of αvβ3 with extracellular matrix ligands such as vitronectin plays a critical role in enhancing IGF1 signaling (Clemmons et al., Growth Horm IGF Res 17(4): 265-70, 2007). It has been reported that inhibiting αvβ3-extracellular matrix interaction (“ligand occupancy”) of αvβ3 inhibited IGF1 actions selectively in cell types that express αvβ3 (Clemmons et al., Growth Horm IGF Res 17(4): 265-70, 2007). Inhibiting “ligand occupancy” of αvβ3 has been reported to block IGF1 induced cell migration (Jones et al., Prog Growth Factor Res 6(2-4): 319-27, 1995), DNA synthesis, IRS-1 phosphorylation, and IGF-1R-linked down stream signaling events, such as activation of PI3K and ERK1/2 (Zheng and Clemmons, Proc Natl Acad Sci USA 95(19): 11217-22, 1998).
The present inventors have demonstrated that expression of αvβ3 enhances proliferation of ovarian cancer cells in the presence of fetal bovine serum (FBS) and in serum-free conditions if IGF1 is present. This indicates that IGF1 is involved in enhanced proliferation of αvβ3-expressing cells. The inventors have demonstrated that αvβ3 binds to IGF1 in several different binding assays. It has been found that three Arg residues at positions 36, 37, and 50 in the C-domain of IGF1 are critical for integrin binding by docking simulation and mutagenesis. Mutation of one or more of these Arg residues to Glu (the R36E/R37E or R50E mutation) effectively reduces integrin binding. Interestingly, the R36E/R37E mutant is defective in inducing cell proliferation and IGF1 intracellular signaling while they still bind to IGF1R. These results suggest that the direct binding to IGF1 plays a role in IGF1 signaling. Notably, the inventors demonstrated that the R36E/R37E mutant as well as the R50E mutant suppressed cell proliferation induced by wild type IGF1, suggesting that these mutants are so-called dominant-negative mutants. Taken together, this discovery indicates that the direct binding of αvβ3 to IGF1 plays a role in IGF1 signaling.
Thus, an IGF1 mutant that does not trigger IGF1 signaling while retaining its ability to bind IGF1R (such as the mutants created by the present inventors) has an immediate utility as a therapeutic in conditions involving inappropriate cellular proliferation, e.g., various forms of cancer. Also, these mutants are a powerful tool for studying the role of integrins in IGF1 signaling. Furthermore, the integrin-binding site within the C-domain of IGF1 provides a valuable tool for identification of additional inhibitors of IGF1-integrin binding, as these inhibitors can be useful in cancer therapy. Because of the prevalence of cancers, there remains a need to develop new strategies for cancer treatment. The present invention addresses this and other related needs.